1. Field of the Invention
The present invention involves transport of complex fluids in channels of nanoscale dimension. More particularly, it examines efficient separation of biomolecular components within complex mixtures, including protein separations.
2. Description of the State of the Art
As nanofluidic devices receive increasing attention, elucidation of phenomena associated with flow of liquids through conduits of nanoscale dimensions remains an important scientific goal. Specifically, the development of chip-based nanofluidic systems for molecular separations (See K. P. Travis, et al., Physical Review E 55 4288 (1997); L. A. Pozhar, K. E. Gubbins, Journal of Chemical Physics 99 8970 (1993)), especially of biomolecular mixtures, based on nanoscale phenomena including entropic trapping (See J. Han, S. W. Turner, H. G. Craighead, Physical Review Letters 86 1394 (1999)), and shear-driven chromatography (See D. Clicq, et al., Journal of Chromatography 979 33 (2002); C. W. Huck, G. Stecher, R. Bakry, G. K. Bonn, Electrophoresis 24 3977 (2003)), has recently received intense interest. In addition to a limited number of experimental efforts (See S. C. Jacobson, J. P. Alarie, J. M. Ramsey, in Proceedings of Micro Total Analysis Systems 2001, Dordrecht, The Netherlands, 57-59 (2001); R. Karlsson, et al., Langmuir 18 4186 (2002); D. Stein, M. Kruithof, C. Dekker, Physical Review Letters 93 035901 (2004)), several important theoretical studies have sought to clarify the effects of nanoconfinement of fluid flows on the molecular distributions and trajectories in electrokinetic flows (See B. J. Loughnane, et al., Journal of Physical Chemistry B 104 5421 (2000); P. J. Kemery, et al., Langmuir 14 2884 (1998); A. P. Thompson, J. Chem. Phys. 119 7503 (2003); Q. S. Pu, J. S. Yun, H. Temkin, S. R. Liu, Nano Letters 4 1099 (2004)). For example, a recent study concluded that effects resulting from the non-continuum nature of electrokinetic flows in nanoscopic pores are associated with varying fluid viscosity very close to the pore wall (See R. Qiao, N. R. Aluru, J. Chem. Phys. 118 4692 (2003)). Further, it was found that with this correction, electroosmotic velocity profiles away from solid surfaces, after a few molecular layers, are in reasonable accordance with the previously elucidated continuum theory (See C. L. Rice and R. Whitehead, J. Phys. Chem. 69 4017(1965); R. J. Hunter, Zeta potential in colloid science: principles and applications. (Academic Press: London, 1981)). Thus far, theoretical investigation of the effects of nanoconfinement on electrokinetic transport of fluids has outpaced experiments because the details of such fluid flows have been unobservable by standard near- and far-field techniques.
Therefore, there has been a long-felt need in the art to (1) develop methodologies for investigation of electrokinetic transport in nanoscale channels using confocal scanning laser microscopy (CSLM), a widely available far field technique; (2) show how data obtained can be compared with analytical models for fluid transport within very small channels; and (3) explore the potential for the use of nanoconfined electrokinetic transport in the development of new methodologies for molecular separation.
With this in mind, one should understand that polyacrylamide gel electrophoresis (PAGE) remains the standard for biomolecular separation and identification in biotechnology. Nevertheless, the set of separation strategies that rely on this technique are hampered by (1) inconvenience and irreproducibility in preparation of the variety of gels needed for the separations, (2) limited resolution and dynamic range of biomolecular separations, (3) susceptibility of the polymer to degradation under high electric fields, (4) limitations in their compatibility with mass spectrometric identification of proteins and (5) relatively large volumes and concentrations of material needed for detection of separated species. Gradient PAGE techniques are recognized to have good resolution and dynamic range, but their utility is greatly hampered by the need for cumbersome gel synthesis protocols and lack of reproducibility.
Previous work on nanofluidic bioseparation systems has included the development of Brownian ratchets (See A. van Oudenaarden et al., Brownian ratchets: Molecular separations in lipid bilayers supported on patterned arrays”, Science, 285, 1046-1048 (1999); C. F. Chou, et al., Sorting by diffusion: An asymmetric obstacle course for continuous molecular separation”, Proc. Natl. Acad. Sci. USA, 96, 13762-13765 (1999)), and entropic traps (See J. Han et al., Separation of long DNA molecules in a microfabricated entropic trap array, Science, 288, 1025, 1029 (2000)), that achieved efficient separation of biomolecules, albeit at rates that cannot be considered commensurate with high throughput technologies. While providing important insight into the behavior of transport of individual molecules (especially DNA) through tortuous nanofluidic systems, these demonstrations have not led to a widespread use of such systems by the biotechnological community. The primary reasons for this is the difficulty by which the nanofluidic systems have been prepared, the high costs of fabrication, and the inability of the fabrication techniques to produce macroscopic arrays of nanofluidic pathways of specified, predetermined, functional design. A primary contribution of this project to the biotechnological world will be to overcome these obstacles by introducing interferometric lithography (IL) as the nanofabrication tool of choice in the fabrication of nanofluidic systems for large scale bioseparations.
To date, the majority of nanofluidic systems have been developed primarily for separation of nucleic acids, while similar systems for separation of other biomolecules lay far behind. Increasingly, microfluidic devices are being developed that have direct application to the burgeoning field of proteomics. (See G. J. M. Bruin, Recent developments in electrokinetically driven analysis on microfabricated devices, Electrophoresis, 21, 3931-3951 (2000)). Analysis of the protein composition of organisms, tissues and single cells under a variety of physiological and environmental conditions is expanding not only our basic understanding of biomolecular function (See N. Anderson, et al., Proteomics: applications in basic and applied biology, Current Opinion in Biotechnology, 11, 408-412 (2000)), but is also showing promise for diverse and tangible rewards in areas such as but not limited to drug discovery (See J. H. Wang et al., Proteomics in drug discovery, Drug Discovery Today, 4, 129-133 (1999)), rapid diagnosis and treatment of disease, and rapid development of vaccines (See R. Aebersold et al., Mass spectrometry in proteomics, Chemical Reviews, 101, 269-295 (2001)), the latter of which is of particular contemporary importance. Proteomic analysis is relying increasingly on precise separation of proteins coupled with the sensitive detection and analysis capabilities of mass spectrometry (See G. Grandi, Antibacterial vaccine design using genomics and proteomics, Trends in Biotechnology, 19, 181-185 (2001)). We believe that our nanostructured devices will be easily integrated into systems such as those being currently developed for interfaces between microfluidic separation devices and chemical detection components.